Human pyruvate dehydrogenase complex (hPDC) plays a gatekeeper[unreadable]s role in the oxidation of pyruvate derived from carbohydrates and the carbon-skeletons of several amino acids, and about 50 percent of daily calorie intake (in term of carbon-flux) is regulated by this enzyme. hPDC is composed of multiple copies of three catalytic components: heterotetrameric (a2[unreadable]2) pyruvate dehydrogenase (PDH), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3); E3-binding protein (BP), and two regulatory enzymes: PDH kinase (PDK, 4 isoenzymes) and phosphatase, (PDP, 2 isoenzymes). Genetic defects in PDC components have illustrated the importance of PDC in energy homeostasis and cause impaired brain development, mental retardation and often early death in children. Reductions in PDC component proteins and activity levels are observed in chronic diseases such as type 2 diabetes, obesity and cardiovascular diseases and also in neurodegenerative diseases such as Alzheimer[unreadable]s disease, Parkinson[unreadable]s disease and Wernicke-Korsakoff syndrome. hPDC has evolved to unique structural organization over prokaryotic PDCs and other members of the a-keto acid dehydrogenase complex family from all species by having BP as additional component and a highly sophisticated mechanism of regulation by multiple isoenzymes of PDKs and PDPs. Recently, we have solved the 3-D structures of human PDH and also of human E3 bound to the E3-binding domain of BP. Based on these developments our three specific aims are to (i) investigate the formation of intermediates in the catalysis of human PDH, (ii) investigate the active site communication in hPDH, and (iii) determine the loci of interactions between hE3 and hBP and also between hE2 and hBP. Site-directed mutagenesis will be employed to introduce desired mutations and human proteins will be over-expressed and purified by affinity chromatography. We will employ the state-of-art instrumentation (such as circular dichroism, 1H NMR, mass spectrometry, rapid stopped-flow circular dichroism, and isothermal titration calorimetry) and chemically synthesized analogs to identify and monitor the intermediates of the PDH reaction. The time-course analysis of the intermediates of PDH reaction will allow us to determine function of individual amino acid residues in catalysis and to characterize the mechanism of communication (flip-flop mechanism) between the two active sites. The roles of specific amino acid residues of hBP and hE3 as well as of hBP and hE2 in protein-protein interactions will be determined by kinetic analysis, gel filtration assay, surface plasmon resonance, isothermal titration calorimetry and ultracentrifugation. The proposed studies combine the extensive expertise of two laboratories to greatly enhance our understanding of the catalytic mechanism of hPDH and the structurefunction relationships of hPDC components and would provide the biochemical basis for some genetic defects in PDC.